CN113881633B - Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells - Google Patents
Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells Download PDFInfo
- Publication number
- CN113881633B CN113881633B CN202111475064.3A CN202111475064A CN113881633B CN 113881633 B CN113881633 B CN 113881633B CN 202111475064 A CN202111475064 A CN 202111475064A CN 113881633 B CN113881633 B CN 113881633B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- hematopoietic stem
- cell
- stem cells
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/145—Thrombopoietin [TPO]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
Abstract
The invention relates to the technical field of dry expansion culture of hematopoietic stem cells, in particular to a culture medium and a method for dry expansion of hematopoietic stem cells of umbilical cord blood in vitro. A culture medium for in-vitro dry expansion of umbilical cord blood hematopoietic stem cells comprises the following components: the cell factor culture medium comprises a basic culture medium, and cell factors and polyvinyl alcohol which are added in the basic culture medium, wherein the cell factors comprise thrombopoietin, stem cell growth factor and FMS-like tyrosine kinase 3 ligand. An in-vitro dry expansion method for umbilical cord blood hematopoietic stem cells comprises the following steps: s1, preparing a culture medium; s2, separating CD34+A cell; s3, amplification culture. The culture medium for dry expansion of hematopoietic stem cells in vitro can obviously improve CD34+CD45RA‑CD90+The amount and proportion of the population of cells expanded in vitro.
Description
Technical Field
The invention relates to the technical field of dry expansion culture of hematopoietic stem cells, in particular to a culture medium and a method for dry expansion of hematopoietic stem cells of umbilical cord blood in vitro.
Background
Hematopoietic Stem Cells (HSCs), the origin of all hematopoietic and immune cells, are a small group of the most primitive hematopoietic cells with high self-renewal and multipotent differentiation potential. Hematopoietic stem cell transplantation is an effective method for treating hematological diseases, hereditary diseases, and immunological diseases. Currently, the main sources of hematopoietic stem cells are bone marrow, peripheral blood, and cord blood. Cord blood-derived hematopoietic stem cells have the following advantages compared to bone marrow and peripheral blood-derived hematopoietic stem cells: wide source, simple collection, no damage to donor and no side effect; the material object is stored, so that the rapid supply can be ensured according to the requirement on time; the placenta barrier has low chance of protecting the infection of pathogenic microorganisms such as viruses and the like, and is relatively pure and safe; the lymphocyte has weak antigenicity and relatively immature function, the requirement on Human Leukocyte Antigen (HLA) matching is low, and the incidence rate and the degree of graft-versus-host disease (GVHD) are low; has higher in vitro proliferation potential and in vivo hematopoietic reconstruction capability. However, the clinical application of cord blood hematopoietic stem cells has the bottleneck problems that the number of single cord blood hematopoietic stem cells is insufficient for large-weight patients and the hematopoietic function recovery time is long. Therefore, expansion of hematopoietic stem cells is required to obtain a sufficient number of hematopoietic stem cells for transplantation.
At present, in vitro expansion of hematopoietic stem cells has been studied for many years, but the optimal in vitro expansion method and system are still not in consensus, and the expansion method and system which can be applied to clinical use are more lacking, and the main reason is that hematopoietic stem cells can be obviously differentiated in the expansion process and cannot be subjected to dry expansion.
CD34+Is a widely recognized surface marker for hematopoietic stem cells, however, CD34+The cells are a heterogeneous population of cells including multipotent progenitors (MPP), short cycle hematopoietic stem cells (ST-HSC) and long cycle hematopoietic stem cells (LT-HSC), wherein the number of self-renewing long cycle hematopoietic stem cells (LT-HSC) determines the success rate of hematopoietic stem cell transplantation. More recently, researchers have identified a particular subpopulation of hematopoietic stem cells: CD34+CD45RA-CD90+Which rebuilds the entire blood system and immune system in vivo after transplantation and continuously maintains the newly formed blood system and immune system (SRadtke, et al
stem cell population for rapid multilineage engraftment in nonhuman primates.SScience relative medicine.2017, DOI: 10.1126/scitranslim. aan 1145), which describes CD34+CD45RA-CD90+The cells are CD34+The cells have a more strongly dry cell population. Therefore, when hematopoietic stem cells are expanded in vitro, CD34 can be preferentially expanded+CD45RA-CD90+Cell population other than CD34+Cells, i.e. CD34+CD45RA-CD90+The expansion of the cell population is higher than that of CD34+The expansion multiple of the cell group, so that the cells after in vitro expansion can better maintain the transplantation capability of the hematopoietic stem cells, the transplantation of the hematopoietic stem cells is facilitated, and the transplantation success rate is improved.
However, most of the hematopoietic stem cell culture media in the prior art are directed to CD34+Cell expansion medium, lack of CD34+CD45RA-CD90+A dry cell population amplification medium and a culture method.
Disclosure of Invention
To solve the above CD34+CD45RA-CD90+The invention provides a culture medium and a method for dry expansion of umbilical cord blood hematopoietic stem cells in vitro.
The technical scheme adopted by the invention for solving the technical problems is as follows: a culture medium for in-vitro dry expansion of umbilical cord blood hematopoietic stem cells comprises the following components: the cell factor-based growth factor culture medium comprises a basic culture medium, and a cell factor and polyvinyl alcohol which are added in the basic culture medium, wherein the cell factor comprises thrombopoietin, stem cell growth factor and FMS-like tyrosine kinase 3 ligand, and the concentration ratio of the thrombopoietin to the stem cell growth factor to the FMS-like tyrosine kinase 3 ligand is (7-14): 1: (7-14).
As an optimization, wherein:
the concentration of the thrombopoietin is 80-110 ng/ml;
the concentration of the stem cell growth factor is 8-11 ng/ml;
the concentration of the FMS-like tyrosine kinase 3 ligand is 80-110 ng/ml;
the concentration of the polyvinyl alcohol is 1-5 mg/ml.
As optimization, the basic culture medium is a serum-free culture medium, and the basic culture medium adopts one of DMEM, IMDM, F12, stemline II and HIPP-T009.
The optimization comprises the following components: serum-free basal medium, 100ng/ml thrombopoietin, 10ng/ml stem cell growth factor, 100ng/ml FMS-like tyrosine kinase 3 ligand and 1mg/ml polyvinyl alcohol.
An in vitro dry expansion method of cord blood hematopoietic stem cells, comprising the culture medium for in vitro dry expansion of cord blood hematopoietic stem cells according to any one of the above, characterized in that: the method comprises the following steps:
s1, preparation of a culture medium: respectively setting final concentrations of thrombopoietin, stem cell growth factor, FMS-like tyrosine kinase 3 ligand and polyvinyl alcohol to be 80-110 ng/ml, 8-11 ng/ml, 80-110 ng/ml and 1-5 mg/ml, setting final addition concentrations of insulin, transferrin, selenium and ethanolamine to be 8-12 mg/L, 4-6 mg/L, 0.005-0.008 mg/L and 1.8-2.2 mg/L, and setting final addition concentration of glutamine to be 280-310 mg/L, so as to obtain a cell culture medium;
s2, separating CD34+Cell: CD34 separated from umbilical cord blood of fresh healthy newborn+A cell;
s3, amplification culture: the CD34 obtained in step S2+The cells are inoculated into the cell culture medium obtained in step S1 and subjected to expansion culture, thereby obtaining expanded hematopoietic stem cells.
As an optimization, the preparation method of the cell culture medium described in step S1 includes the following steps:
a1, preparing stock solution: preparing the sterile freeze-dried powder of thrombopoietin, stem cell growth factor and FM-like tyrosine kinase 3 ligand into stock solutions of 15mg/ml, 1mg/ml and 15mg/ml by using a serum-free culture medium respectively for later use; preparing 100mg/ml of polyvinyl alcohol with ultrapure water, and filtering and sterilizing the polyvinyl alcohol in an ultra-clean bench by using a disposable sterile filter head of 0.45 mu m for later use;
a2, preparation of culture medium: and B, respectively adding the stock solution prepared in the step A1, polyvinyl alcohol, insulin, transferrin, selenium, ethanolamine and glutamine into a serum-free basal medium to ensure that the final concentrations of thrombopoietin, stem cell growth factor, FMS-like tyrosine kinase 3 ligand and polyvinyl alcohol are respectively 80-110 ng/ml, 8-11 ng/ml, 80-110 ng/ml and 1-5 mg/ml, and the final addition concentrations of insulin, transferrin, selenium, ethanolamine and glutamine are respectively 8-12 mg/L, 4-6 mg/L, 0.005-0.008 mg/L, 1.8-2.2 mg/L and 280-310 mg/L.
Preferably, the amplification culture conditions in step S3 are 37 ℃ and CO2The concentration was 5%.
Preferably, the time of the amplification culture in the step S3 is 3-7 days.
As optimization, CD34 in step S2+The method for separating the cells comprises the following steps:
b1, pretreatment of cord blood: injecting 100ml of a fresh healthy newborn umbilical cord blood sample for providing hematopoietic stem cells to be amplified into a 250ml centrifuge tube under an aseptic condition, centrifuging at room temperature of 1200rpm for 10 minutes, removing upper plasma, adding physiological saline to dilute blood cell precipitates to 2 times of the original volume, and uniformly blowing and stirring by using a pipette to obtain a blood cell suspension;
b2, centrifugation of blood cell suspension: slowly loading the blood cell suspension obtained by the treatment in the step B1 on a prepared lymphocyte separation liquid with 0.4-time volume, keeping the layering clear, centrifuging at room temperature of 2000rpm for 20 minutes, dividing the centrifugal tube into four layers due to different densities, wherein the uppermost layer is saline and plasma, the lower layer is boundary layer cells mainly comprising lymphocytes and monocytes, the third layer is the lymphocyte separation liquid, and the fourth layer is precipitated red blood cells and granulocytes;
b3, isolation of mononuclear cells: b, absorbing most of the uppermost layer of the blood cell suspension processed in the step B2 by using a pipette, discarding the most of the liquid, transferring the boundary cell layer of the second layer into a 50ml centrifuge tube, wherein the cell suspension of each tube does not exceed 25ml, adding physiological saline into the cell suspension to supplement the volume of the cell suspension to 50ml, uniformly mixing, centrifuging at room temperature of 1200rpm for 10 minutes, discarding the supernatant after centrifugation, adding the physiological saline into the cell suspension for re-suspension, centrifuging at room temperature of 1200rpm for 10 minutes after uniform mixing, discarding the supernatant, and obtaining mononuclear cells;
b4, isolated CD34+Hematopoietic stem cells: adopting 50 mu l of human CD34 for the mononuclear cells obtained in the step B3+Resuspending a mixed solution of magnetic beads, 50 mul of blocking solution and 150 mul of 0.5% bovine serum albumin, and incubating for 30min at 4 ℃; adding 10ml of 0.01mol/L phosphate buffer solution after incubation is finished, centrifuging at 1500rpm for 10 minutes after uniform mixing, discarding supernatant, resuspending 500 microliter of 0.5% bovine serum albumin, transferring to a separation column which is placed on a magnet and is rinsed by the 0.01mol/L phosphate buffer solution, when liquid completely flows out, washing the separation column for 2 times by using 500 microliter of 0.5% bovine serum albumin, placing the separation column in a collecting pipe after washing is finished, adding 1ml of 0.5% bovine serum albumin, pushing the liquid into the collecting pipe by using a plunger piston, and obtaining CD34+And (5) counting hematopoietic stem cells.
The culture medium and the method for in-vitro dry expansion of the umbilical cord blood hematopoietic stem cells have the advantages that:
(1) by adding the cytokines thrombopoietin, stem cell growth factor and FMS-like tyrosine kinase 3 ligand into the culture medium and controlling the proportion of the cytokines thrombopoietin, the stem cell growth factor and the FMS-like tyrosine kinase 3 ligand, the concentration of the thrombopoietin and the FMS-like tyrosine kinase 3 ligand is the same and is far greater than the concentration (7-14 times) of the stem cell growth factor, and the effect of the growth factor on hematopoietic stem cells CD34 can be achieved+CD45RA-CD90+Specific culture of cell population to make CD34+CD45RA-CD90+The expansion of the cell population is much higher than that of CD34+Fold expansion of the cells;
(2) the thrombopoietin, the stem cell growth factor and the FMS-like tyrosine kinase 3 ligand with specific concentration proportion have synergistic effect, can promote the expansion of hematopoietic stem cells, avoid the over-differentiation of the hematopoietic stem cells, and can obviously improve the hematopoietic stem cells CD34+CD45RA-CD90+The number and proportion of in vitro expansion of the cell population;
(3) by using the serum-free basal medium and adding the polyvinyl alcohol, on one hand, the serum-free medium eliminates the adverse effect of uncontrollable components and serum albumin in serum production on the differentiation of the hematopoietic stem cells, and on the other hand, the addition of the polyvinyl alcohol makes up the change of the deletion of the serum albumin in the medium on the physical environment required by cell growth, so that the hematopoietic stem cells can be expanded in the environment close to human blood without over-differentiation;
(4) hematopoietic stem cell CD34 expanded by the culture medium of the present application+CD45RA-CD90+Population ratio of cells CD34+The cell population has higher expansion multiple, which shows that the expansion culture medium and the method provided by the invention have the advantages of dry expansion of hematopoietic stem cells and have stronger potential for reconstructing a hematopoietic system and an immune system.
Drawings
FIG. 1 is a graph showing the results of in vitro amplification of hematopoietic stem cell samples of example 1 in a medium provided by the present invention.
FIG. 2 is a graph showing the amplification results obtained in the in vitro amplification process of hematopoietic stem cell samples of example 2 in the medium provided by the present invention.
FIG. 3 is a graph showing the results of in vitro amplification of hematopoietic stem cell samples of example 3 in a medium provided by the present invention.
FIG. 4 is a graph showing the results of the expansion of the hematopoietic stem cell sample of comparative example 1.
FIG. 5 is a graph showing the results of expansion of a hematopoietic stem cell sample of comparative example 2.
Wherein the abscissa is the culture time, the left ordinate is the cell number, the right ordinate is the cell expansion fold, and the unit of the cell number is 104Per ml, black bars are CD34+Cell mass, white bars are CD34+CD45RA-CD90+Cell mass, solid black line CD34+Fold cell expansion, dashed black line CD34+CD45RA-CD90+Fold expansion of cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings or the orientations or positional relationships that the products of the present invention are conventionally placed in use, and are only used for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like are used solely to distinguish one from another and are not to be construed as indicating or implying relative importance.
Example 1:
100ml of a fresh healthy newborn umbilical cord blood sample for providing hematopoietic stem cells to be amplified is injected into a 250ml centrifuge tube under the aseptic condition, the centrifuge tube is centrifuged at room temperature and 1200rpm for 10 minutes, the upper plasma layer is discarded, physiological saline is added to dilute the blood cell sediment to 2 times of the original volume, and the mixture is blown by a pipette and uniformly mixed.
The blood cell suspension was slowly loaded onto a pre-prepared 0.4-fold volume of lymphocyte separation medium, taking care to keep the layering clear. Centrifuging at 2000rpm for 20min at room temperature, wherein the centrifugal tube is divided into four layers due to different densities, and the uppermost layer is saline and plasma; the next layer is boundary layer cells, which mainly comprise lymphocytes and monocytes, the third layer is lymphocyte separation liquid, and the fourth layer is precipitated red blood cells and granulocytes.
Sucking most of the liquid on the uppermost layer by using a pipette, discarding, transferring the boundary cell layer on the second layer into a 50ml centrifuge tube, adding physiological saline into the cell suspension to supplement 50ml, uniformly mixing, and centrifuging at room temperature of 1200rpm for 10 minutes, wherein the cell suspension in each tube does not exceed 25 ml; centrifuging, removing supernatant, adding physiological saline for resuspension, mixing uniformly, centrifuging at room temperature of 1200rpm for 10 minutes, and removing supernatant to obtain mononuclear cells;
adopting 50 mu l of human CD34 to the obtained mononuclear cells+Resuspending a mixed solution of magnetic beads, 50 mul of blocking solution and 150 mul of 0.5% bovine serum albumin, and incubating for 30min at 4 ℃; after the incubation is finished, 10ml of 0.01mol/L phosphate buffer solution is added, the mixture is centrifuged at 1500rpm for 10 minutes after being uniformly mixed, the supernatant is discarded, 500 mu L of 0.5% bovine serum albumin is resuspended, the mixture is transferred to a separation column which is arranged on a magnet and is rinsed by the 0.01mol/L phosphate buffer solution, after the liquid completely flows out, the separation column is arranged in a collecting pipe after the 500 mu L of 0.5% bovine serum albumin is washed for 2 times, 1ml of 0.5% bovine serum albumin is added, the liquid is pushed into the collecting pipe by a plunger, and CD34 is obtained+And (5) counting hematopoietic stem cells.
Subjecting the cord blood sample of fresh healthy newborn to the above steps to obtain CD34+The cell is inoculated in a cell culture medium for amplification culture, the cell culture medium adopts a serum-free medium IMDM culture medium, the thrombopoietin concentration is added to 81.2ng/ml, the stem cell growth factor is added to the concentration of 10.9ng/ml, the FMS-like tyrosine kinase 3 ligand is added to the concentration of 80.5ng/ml, the polyvinyl alcohol is added to the concentration of 3mg/ml, the insulin, the transferrin, the selenium and the ethanolamine are added to the final concentrations of 9mg/L, 5.2mg/L, 0.0069mg/L and 2.1mg/L respectively, and the glutamine is added to the final concentration of 310 mg/L.
The cell inoculation amount in the 24-well plate is 5 multiplied by 104Per ml, 5% CO at 37 ℃2Culturing in an incubator, and sampling and detecting on 3 rd, 5 th and 7 th days.
Example 2:
will be used to provide a nucleic acid sequence to be amplifiedFresh healthy neonatal cord blood sample of hematopoietic stem cells the same CD34 as in example 1 was used+Separating cells, and processing to obtain CD34+And (5) counting hematopoietic stem cells.
Subjecting the cord blood sample of fresh healthy newborn to the above steps to obtain CD34+The cell is inoculated in a cell culture medium for amplification culture, the cell culture medium adopts a serum-free culture medium F12 culture medium, the concentration of thrombopoietin is added to 109ng/ml, the concentration of stem cell growth factor is added to 8.2ng/ml, the concentration of FMS-like tyrosine kinase 3 ligand is added to 110ng/ml, the concentration of polyvinyl alcohol is added to 5mg/ml, insulin, transferrin, selenium and ethanolamine are added to final concentrations of 11.8mg/L, 4.9mg/L, 0.0062mg/L and 2.1mg/L respectively, and glutamine is added to a final concentration of 289 mg/L.
The cell inoculation amount in the 24-well plate is 5 multiplied by 104Per ml, 5% CO at 37 ℃2Culturing in an incubator, and sampling and detecting on 3 rd, 5 th and 7 th days.
Example 3:
the same CD34 as in example 1 was used for cord blood samples of fresh healthy newborn infants for providing hematopoietic stem cells to be expanded+Separating cells, and processing to obtain CD34+And (5) counting hematopoietic stem cells.
Subjecting the cord blood sample of fresh healthy newborn to the above steps to obtain CD34+The cell is inoculated in a cell culture medium for amplification culture, the cell culture medium adopts a serum-free medium HIPP-T009 medium, thrombopoietin concentration is added to 101ng/ml, stem cell growth factor is added to a concentration of 10.3ng/ml, FMS-like tyrosine kinase 3 ligand is added to a concentration of 102.1ng/ml, polyvinyl alcohol is added to 1mg/ml, insulin, transferrin, selenium and ethanolamine are added to final concentrations of 10mg/L, 5.5mg/L, 0.0067mg/L and 2.0mg/L respectively, and glutamine is added to a final concentration of 292 mg/L.
The cell inoculation amount in the 24-well plate is 5 multiplied by 104Per ml, 5% CO at 37 ℃2Culturing in an incubator, and sampling and detecting on 3 rd, 5 th and 7 th days.
Comparative example 1:
will be used forCord blood sample of fresh healthy newborn provided with hematopoietic stem cells to be expanded using CD34 same as in example 1+Separating cells, and processing to obtain CD34+And (5) counting hematopoietic stem cells.
Subjecting the cord blood sample of fresh healthy newborn to the above steps to obtain CD34+The cells are inoculated in a cell culture medium for amplification culture, the cell culture medium adopts a serum-free medium HIPP-T009 medium, thrombopoietin concentration is added to 21.5ng/ml, stem cell growth factor is added to the concentration of 20ng/ml, FMS-like tyrosine kinase 3 ligand is added to the concentration of 19.6ng/ml, polyvinyl alcohol is added to the concentration of 2mg/ml, insulin, transferrin, selenium and ethanolamine are added to the final concentrations of 8.5mg/L, 5.3mg/L, 0.0071mg/L and 2.1mg/L respectively, and glutamine is added to the final concentration of 299 mg/L.
The cell inoculation amount in the 24-well plate is 5 multiplied by 104Per ml, 5% CO at 37 ℃2Culturing in an incubator, and sampling and detecting on 3 rd, 5 th and 7 th days.
Comparative example 2:
the same CD34 as in example 1 was used for cord blood samples of fresh healthy newborn infants for providing hematopoietic stem cells to be expanded+Separating cells, and processing to obtain CD34+And (5) counting hematopoietic stem cells.
Subjecting the cord blood sample of fresh healthy newborn to the above steps to obtain CD34+The cell is inoculated in a cell culture medium for amplification culture, the cell culture medium adopts a serum-free medium IMDM culture medium, the thrombopoietin concentration is added to 98ng/ml, the stem cell growth factor is added to the concentration of 80ng/ml, the FMS-like tyrosine kinase 3 ligand is added to the concentration of 22ng/ml, the polyvinyl alcohol is added to 3mg/ml, the insulin, the transferrin, the selenium and the ethanolamine are added to the final concentrations of 9.4mg/L, 4.8mg/L, 0.0068mg/L and 1.9mg/L respectively, and the glutamine is added to the final concentration of 289 mg/L.
The cell inoculation amount in the 24-well plate is 5 multiplied by 104Per ml, 5% CO at 37 ℃2Culturing in an incubator, and sampling and detecting on 3 rd, 5 th and 7 th days.
The sampling detection method for stem cells designed in examples 1 to 3 and comparative examples 1 to 2 includes the following steps:
(1) CD34 obtained from mononuclear cells+Taking 10 mu l of cell suspension of the cells or the cell suspension after the amplification culture to count the cells;
(2) adding another 10 mu L of phosphate buffer solution of 0.01mol/L to 100 mu L, and respectively adding the CD90 marked by the PE into the mixture+Antibody, FITC-labeled CD34+Antibody and APC labeled CD45RA -3 mul of each antibody is incubated for 20min in a dark place,
(3) and (3) adding 100 mu l of buffer solution into the solution obtained in the step (2), and detecting the cell phenotype by using a flow cytometer.
(4) Detection of CD34 by cytometry before and after amplification+Cells and CD34+CD45RA-CD90+The total cell count and CD34 were calculated from the cell ratio+Fold expansion of cells and CD34+CD45RA-CD90+Fold expansion of cells.
The CD34 of examples 1-3 and comparative examples 1-2 was calculated by the sampling detection method+Cells and CD34+CD45RA-CD90+The results of the culture of the cell population were as follows:
table 1: example 1 culture results
Table 2: example 2 culture results
Table 3: example 3 culture results
Table 4: comparative example 1 culture results
Table 5: comparative example 2 culture results
The CD34 of examples 1-3 and comparative examples 1-2 was calculated by the sampling detection method+Cells and CD34+CD45RA-CD90+The highest fold expansion of the cell population is as follows:
table 6: CD34 of examples 1-3 and comparative examples 1-2+Cells and CD34+CD45RA-CD90+Highest fold expansion of cell population
| Item | CD34+Fold expansion of cells | CD34+CD45RA-CD90+Fold expansion of cell population |
| Example 1 | 3.14 times of | 7.78 times of |
| Example 2 | 3.65 times of | 14.00 times of |
| Example 3 | 5.57 times of | 17.90 times of |
| Comparative example 1 | 21.62 times of | 9.70 times of |
| Comparative example 2 | 14.34 times of | 6.61 times of |
From the culture results of the above examples and comparative examples, it is understood that CD34 with higher dryness can be preferentially amplified by the culture medium and culture method of the present application+CD45RA-CD90+The cell population can obviously increase the number and proportion of dry cells obtained by in vitro expansion of hematopoietic stem cells.
CD34 is well known to researchers in the hematopoietic stem cell field+Is a widely recognized surface marker for hematopoietic stem cells, however, CD34+Cells are a heterogeneous population of cells in which the number of long-cycle hematopoietic stem cells with self-renewal capacity determines the success rate of hematopoietic stem cell transplantation. In addition, researchers have identified a particular subpopulation of hematopoietic stem cells, CD34+CD45RA-CD90+Cell populations that reconstitute the entire blood system and immune system in vivo after transplantation and sustain the newly formed blood system and immune system, demonstrate CD34+CD45RA-CD90+Dryness ratio of cells CD34+And is stronger. The hematopoietic stem cells expanded in this patent are CD34+CD45RA-CD90+Population ratio of cells CD34+The cell population has higher expansion multiple, which shows that the expansion culture medium and the method provided by the invention have the advantages of dry expansion of hematopoietic stem cells and have stronger potential for reconstructing a hematopoietic system and an immune system.
The above embodiments are only specific cases of the present invention, and the scope of the present invention includes but is not limited to the product forms and forms of the above embodiments, and any medium and method for dry expansion of cord blood hematopoietic stem cells in vitro according to the claims of the present invention and any suitable changes or modifications thereof by one of ordinary skill in the art are intended to fall within the scope of the present invention.
Claims (6)
1. A culture medium for dry expansion of umbilical cord blood hematopoietic stem cells in vitro is characterized in that: the cell culture medium consists of a basic culture medium, and cytokines, polyvinyl alcohol, insulin, transferrin, selenium, ethanolamine and glutamine which are added in the basic culture medium;
the cytokine consists of thrombopoietin, stem cell growth factor and FMS-like tyrosine kinase 3 ligand, wherein the concentration ratio of the thrombopoietin to the stem cell growth factor to the FMS-like tyrosine kinase 3 ligand is (7-14): 1: (7-14), the concentration of thrombopoietin is 80-110 ng/ml, the concentration of stem cell growth factor is 8-11 ng/ml, the concentration of FMS-like tyrosine kinase 3 ligand is 80-110 ng/ml, and the concentration of polyvinyl alcohol is 1-5 mg/ml;
wherein the final addition concentrations of insulin, transferrin, selenium and ethanolamine are 8-12 mg/L, 4-6 mg/L, 0.005-0.008 mg/L and 1.8-2.2 mg/L, and the final addition concentration of glutamine is 280-310 mg/L;
the basic culture medium is a serum-free culture medium, and adopts one of DMEM, IMDM, F12, stemlineII and HIPP-T009.
2. An in vitro dry expansion method of cord blood hematopoietic stem cells, which adopts the culture medium for in vitro dry expansion of cord blood hematopoietic stem cells according to claim 1, and is characterized in that: the method comprises the following steps:
s1, preparation of a culture medium: respectively setting final concentrations of thrombopoietin, stem cell growth factor, FMS-like tyrosine kinase 3 ligand and polyvinyl alcohol to be 80-110 ng/ml, 8-11 ng/ml, 80-110 ng/ml and 1-5 mg/ml, setting final addition concentrations of insulin, transferrin, selenium and ethanolamine to be 8-12 mg/L, 4-6 mg/L, 0.005-0.008 mg/L and 1.8-2.2 mg/L, and setting final addition concentration of glutamine to be 280-310 mg/L, so as to obtain a cell culture medium;
s2, separating CD34+Cell: CD34 separated from umbilical cord blood of fresh healthy newborn+A cell;
s3, amplification culture: the CD34 obtained in step S2+The cells are inoculated into the cell culture medium obtained in step S1 and subjected to expansion culture, thereby obtaining expanded hematopoietic stem cells.
3. The method for dry expansion of cord blood hematopoietic stem cells in vitro according to claim 2, wherein: the preparation method of the cell culture medium described in step S1 includes the steps of:
a1, preparing stock solution: preparing the sterile freeze-dried powder of thrombopoietin, stem cell growth factor and FM-like tyrosine kinase 3 ligand into stock solutions of 15mg/ml, 1mg/ml and 15mg/ml by using a serum-free culture medium respectively for later use;
preparing 100mg/ml of polyvinyl alcohol with ultrapure water, and filtering and sterilizing the polyvinyl alcohol in an ultra-clean bench by using a disposable sterile filter head of 0.45 mu m for later use;
a2, preparation of culture medium: and B, respectively adding the stock solution, polyvinyl alcohol, insulin, transferrin, selenium, ethanolamine and glutamine prepared in the step A1 into a serum-free basal medium to ensure that the final concentrations of thrombopoietin, stem cell growth factor, FMS-like tyrosine kinase 3 ligand and polyvinyl alcohol are respectively 80-110 ng/ml, 8-11 ng/ml, 80-110 ng/ml and 1-5 mg/ml, the final addition concentrations of insulin, transferrin, selenium and ethanolamine are respectively 8-12 mg/L, 4-6 mg/L, 0.005-0.008 mg/L and 1.8-2.2 mg/L, and the final addition concentration of glutamine is 280-310 mg/L, so as to obtain the cell culture medium.
4. The method for dry expansion of cord blood hematopoietic stem cells in vitro according to claim 3, wherein: the conditions for amplification culture in the step S3 are 37 ℃ and CO2The concentration was 5%.
5. The method for dry expansion of cord blood hematopoietic stem cells in vitro according to claim 4, wherein: the time of amplification culture in the step S3 is 3-7 days.
6. The method for dry expansion of cord blood hematopoietic stem cells in vitro according to claim 5, wherein: CD34 in step S2+The method for separating the cells comprises the following steps:
b1, pretreatment of cord blood: injecting 100ml of a fresh healthy newborn umbilical cord blood sample for providing hematopoietic stem cells to be amplified into a 250ml centrifuge tube under an aseptic condition, centrifuging at room temperature of 1200rpm for 10 minutes, removing upper plasma, adding physiological saline to dilute blood cell precipitates to 2 times of the original volume, and uniformly blowing and stirring by using a pipette to obtain a blood cell suspension;
b2, centrifugation of blood cell suspension: slowly loading the blood cell suspension obtained by the treatment in the step B1 on a prepared lymphocyte separation liquid with 0.4-time volume, keeping the layering clear, centrifuging at room temperature of 2000rpm for 20 minutes, and separating the centrifuge tube into four layers due to different densities, wherein the uppermost layer is saline and plasma, the lower layer is boundary layer cells mainly comprising lymphocytes and monocytes, the third layer is the lymphocyte separation liquid, and the fourth layer is precipitated red blood cells and granulocytes;
b3, isolation of mononuclear cells: b, absorbing most of the uppermost layer of the blood cell suspension processed in the step B2 by using a pipette, discarding the most of the liquid, transferring the boundary cell layer of the second layer into a 50ml centrifuge tube, wherein the cell suspension of each tube does not exceed 25ml, adding physiological saline into the cell suspension to supplement the volume of the cell suspension to 50ml, uniformly mixing, centrifuging at room temperature of 1200rpm for 10 minutes, discarding the supernatant after centrifugation, adding the physiological saline into the cell suspension for re-suspension, centrifuging at room temperature of 1200rpm for 10 minutes after uniform mixing, discarding the supernatant, and obtaining mononuclear cells;
b4, isolated CD34+Hematopoietic stem cells: adopting 50 mu l of human CD34 for the mononuclear cells obtained in the step B3+Resuspending a mixed solution of magnetic beads, 50 mul of blocking solution and 150 mul of 0.5% bovine serum albumin, and incubating for 30min at 4 ℃; adding 10ml of 0.01mol/L phosphate buffer solution after the incubation is finished, centrifuging at 1500rpm for 10 minutes after the uniform mixing, discarding the supernatant, and resuspending 500 mul of 0.5% bovine serum albuminTransferring the solution to a separation column which is arranged on a magnet and is rinsed by 0.01mol/L phosphate buffer solution, when the solution completely flows out, washing the separation column for 2 times by using 500 mul 0.5% bovine serum albumin, arranging the separation column in a collecting pipe after the washing is finished, adding 1ml 0.5% bovine serum albumin, pushing the solution into the collecting pipe by using a plunger piston, and obtaining CD34+And (5) counting hematopoietic stem cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111475064.3A CN113881633B (en) | 2021-12-06 | 2021-12-06 | Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111475064.3A CN113881633B (en) | 2021-12-06 | 2021-12-06 | Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113881633A CN113881633A (en) | 2022-01-04 |
| CN113881633B true CN113881633B (en) | 2022-02-22 |
Family
ID=79015638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111475064.3A Active CN113881633B (en) | 2021-12-06 | 2021-12-06 | Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113881633B (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101310007A (en) * | 2005-04-19 | 2008-11-19 | 约翰·霍普金斯大学 | Method of using stroma cells from cord blood to expand and engraft nucleated cells from cord blood |
| CN102080065A (en) * | 2010-12-09 | 2011-06-01 | 协和干细胞基因工程有限公司 | Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof |
| MX2010014281A (en) * | 2010-12-20 | 2012-06-20 | Itesm | Culture protocol for the expansion of human hematopoietic stem cells (cd133+ and cd34+) obtained from leukapheresis. |
| CN105112374A (en) * | 2015-09-13 | 2015-12-02 | 广州暨南生物医药研究开发基地有限公司 | Ex-vivo expansion culture medium of umbilical cord blood hematopoietic stem cells and application thereof |
| CN105713879A (en) * | 2014-12-01 | 2016-06-29 | 顺昊细胞生物技术（天津）股份有限公司 | Culture system for umbilical cord blood hematopoietic stem cell amplification and application thereof |
| CN107034189A (en) * | 2017-05-31 | 2017-08-11 | 东莞市保莱生物科技有限公司 | A kind of candidate stem cell cultural method |
| CN107630002A (en) * | 2017-11-03 | 2018-01-26 | 广州市天河诺亚生物工程有限公司 | A kind of amplification method of umbilical cord blood hematopoietic stem cell |
| CN109207427A (en) * | 2018-09-17 | 2019-01-15 | 诺未科技（北京）有限公司 | A method of hematopoietic progenitor cells are changed into candidate stem cell |
| CN109207426A (en) * | 2018-09-17 | 2019-01-15 | 诺未科技（北京）有限公司 | A method of hematopoietic progenitor cells are changed into candidate stem cell |
| CN109593717A (en) * | 2018-12-29 | 2019-04-09 | 广州和能生物科技有限公司 | A kind of stem cell serum-free culture medium |
| CN110628718A (en) * | 2019-09-29 | 2019-12-31 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Stem cell amplification culture medium and stem cell culture method |
| CN110951686A (en) * | 2019-11-14 | 2020-04-03 | 武汉济源高科技有限公司 | Hematopoietic stem cell in-vitro amplification culture system and method |
| WO2020089411A1 (en) * | 2018-10-31 | 2020-05-07 | Mesoblast International Sarl | Expansion of hematopoietic stem cells |
| CN113151172A (en) * | 2021-05-14 | 2021-07-23 | 郑州优倍得生物科技有限公司 | Culture medium for amplifying umbilical cord blood hematopoietic stem cells |
-
2021
- 2021-12-06 CN CN202111475064.3A patent/CN113881633B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101310007A (en) * | 2005-04-19 | 2008-11-19 | 约翰·霍普金斯大学 | Method of using stroma cells from cord blood to expand and engraft nucleated cells from cord blood |
| CN102080065A (en) * | 2010-12-09 | 2011-06-01 | 协和干细胞基因工程有限公司 | Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof |
| MX2010014281A (en) * | 2010-12-20 | 2012-06-20 | Itesm | Culture protocol for the expansion of human hematopoietic stem cells (cd133+ and cd34+) obtained from leukapheresis. |
| CN105713879A (en) * | 2014-12-01 | 2016-06-29 | 顺昊细胞生物技术（天津）股份有限公司 | Culture system for umbilical cord blood hematopoietic stem cell amplification and application thereof |
| CN105112374A (en) * | 2015-09-13 | 2015-12-02 | 广州暨南生物医药研究开发基地有限公司 | Ex-vivo expansion culture medium of umbilical cord blood hematopoietic stem cells and application thereof |
| CN107034189A (en) * | 2017-05-31 | 2017-08-11 | 东莞市保莱生物科技有限公司 | A kind of candidate stem cell cultural method |
| CN107630002A (en) * | 2017-11-03 | 2018-01-26 | 广州市天河诺亚生物工程有限公司 | A kind of amplification method of umbilical cord blood hematopoietic stem cell |
| CN109207427A (en) * | 2018-09-17 | 2019-01-15 | 诺未科技（北京）有限公司 | A method of hematopoietic progenitor cells are changed into candidate stem cell |
| CN109207426A (en) * | 2018-09-17 | 2019-01-15 | 诺未科技（北京）有限公司 | A method of hematopoietic progenitor cells are changed into candidate stem cell |
| WO2020089411A1 (en) * | 2018-10-31 | 2020-05-07 | Mesoblast International Sarl | Expansion of hematopoietic stem cells |
| CN109593717A (en) * | 2018-12-29 | 2019-04-09 | 广州和能生物科技有限公司 | A kind of stem cell serum-free culture medium |
| CN110628718A (en) * | 2019-09-29 | 2019-12-31 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Stem cell amplification culture medium and stem cell culture method |
| CN110951686A (en) * | 2019-11-14 | 2020-04-03 | 武汉济源高科技有限公司 | Hematopoietic stem cell in-vitro amplification culture system and method |
| CN113151172A (en) * | 2021-05-14 | 2021-07-23 | 郑州优倍得生物科技有限公司 | Culture medium for amplifying umbilical cord blood hematopoietic stem cells |
Non-Patent Citations (1)
| Title |
|---|
| A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates;Stefan Radtke等;《SCIENCE TRANSLATIONAL MEDICINE》;20171101;第1-10页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113881633A (en) | 2022-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6221576B1 (en) | Process for preparing macrophages, kits, and compositions for the use of this process | |
| CN109628397B (en) | Method for in-vitro amplification culture of NK (natural killer) cells | |
| CN111454903B (en) | Immune cell in vitro culture, induction, activation and cryopreservation method and cell bank establishment thereof | |
| CN114134114B (en) | Method for amplifying natural killer cells from placenta tissue | |
| CN112608896A (en) | NK cell culture method and application thereof | |
| JPH08511935A (en) | Selective cell growth | |
| JPH0819157B2 (en) | Serum-free and mitogen-free T cell growth factor and method for producing the same | |
| Wong et al. | Development of a closed-system process for clinical-scale generation of DCs: evaluation of two monocyte-enrichment methods and two culture containers | |
| CN101182488A (en) | New use of mesenchyma stem cell | |
| CN111548994B (en) | Cell culture medium and method for culturing NK cells by using same | |
| CN112080469B (en) | Application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro | |
| CN109182263A (en) | A method of palace film mescenchymal stem cell is separated with Tea Saponin dissolution menstruation erythrocyte | |
| CN108841790A (en) | A kind of method of the mononuclearcell induction CIK cell in placenta source | |
| CN113881633B (en) | Culture medium and method for in-vitro dry amplification of umbilical cord blood hematopoietic stem cells | |
| CN110862962A (en) | Method for culturing and amplifying NK cells in vitro by using gallic acid | |
| CN110857435B (en) | Culture medium for culturing immune cells separated from cord blood and culture method thereof | |
| CN107090434B (en) | Blood anticoagulation and preservation method for improving CIK cell culture effect | |
| CN113481157B (en) | Optimized preparation method of specific antiviral adoptive immune cells | |
| CN115197909B (en) | NK cell in-vitro culture method | |
| CN114807031A (en) | Construction method of human peripheral blood immune cell bank and stem cell bank | |
| CN111690607B (en) | Efficient killer cell in-vitro culture kit and culture method | |
| CN111154721B (en) | NK cell amplification method | |
| CN114686432B (en) | Efficient amplification culture system for neutrophils and application thereof | |
| CN113512529B (en) | Preparation method of specific antiviral adoptive immune cell AB | |
| CN116396934B (en) | Culture method of cord blood Treg cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |